Reasoning that proteins that physically associate with the osmoprotective transcription factor TonEBP/OREBP/NFAT5 (TonEBP) are likely to regulate or support its activity we used proteomics to identify them. We stably expressed amino acids 1-547 of TonEBP in HEK 293 cells and immunoprecipitated it plus associated proteins from the nuclei of cells exposed to high NaCl, thereby identifying 15 associated proteins. The associated proteins fall into several classes: 1) DNA-dependent protein kinase, both its catalytic subunit and regulatory subunit, Ku86, and MDC1;2) RNA helicases, namely RNA helicase A, nucleolar RNA helicase II/Gu, and DEAD-box RNA helicase p72;3) small or heterogeneous nuclear ribonucleoproteins (snRNPs or hnRNPs), namely U5 snRNP-specific 116 kDa protein, U5 snRNP-specific 200 kDa protein, hnRNP U, hnRNP M, hnRNP K, and hnRNP F;4) heat shock proteins, namely Hsp90beta and Hsc70;and 5) poly(ADP-ribose) polymerase-1 (PARP-1). We confirmed identification of most of the proteins by Western analysis and also demonstrated by electrophoretic mobility-shift assay that they are present in the large complex that binds specifically along with TonEBP/OREBP to its cognate DNA element. In addition, we found that PARP-1, MDC1 and Hsp90 modulate TonEBP/OREBP activity. PARP-1 expression reduces TonEBP/OREBP transcriptional activity and the activity of its transactivating domain. Hsp90 and MDC1 enhance those activities and sustain the increased abundance of TonEBP/OREBP protein in cells exposed to high NaCl. We are currently extending these proteomic studies to identify additional proteins that associate with TonEBP-1-1531 and also to identify amino acids in TonEBP that are phosphorylated by high salt. Until now we have identified several phosphorylated amino acids and have confirmed with phospho-specific antibodies that phosphorylation of some of them is osmotically regulated. Also, mutation of those amino acids affects osmotic regulation of TonEBP. Glycerophosphocholine (GPC) is an osmoprotective compatible and counteracting organic osmolyte that accumulates in renal inner medullary cells in response to high NaCl and urea. We previously found that high NaCl and/or urea increases GPC in renal (Madin-Darby canine kidney, MDCK) cells and that the GPC is derived from phosphatidylcholine, catalyzed by a phospholipase that was not identified at that time. Neuropathy target esterase (NTE) was recently shown to be a phospholipase B that catalyzes production of GPC from phosphatidylcholine. Therefore, we tested whether NTE contributes to the high NaCl-induced increase of GPC synthesis in renal cells, finding that it does. In mouse inner medullary collecting duct (mIMCD3) cells, high NaCl increases NTE mRNA and protein. Diisopropyl fluorophosphate, which inhibits NTE esterase activity, reduces GPC accumulation, as does an siRNA that specifically reduces NTE protein abundance. The 20-h half-life of NTE mRNA is unaffected by high NaCl, but knockdown of TonEBP by a specific siRNA inhibits the high NaCl-induced increase of NTE mRNA. Further, the lower renal inner medullary interstitial NaCl concentration that occurs chronically in ClCK1-/- mice and acutely in normal mice given furosemide is associated with lower NTE mRNA and protein. Thus, high NaCl increases transcription of NTE, mediated by TonEBP, and the resultant increase of NTE expression contributes to increased production and accumulation of GPC in mammalian renal cells in tissue culture and in vivo. We previously found that high urea and/or NaCl inhibit the activity of a phosphodiesterase (GPC-PDE) that catalyzes breakdown of GPC to choline and glycerol phosphate, and that this contributes to osmotic induction of GPC. We have identified the phosphodiesterase as Gdpd5. Recombinant Gdpd5 immunoprecipitated from mIMCD3 cells has GPC-PDE activity and the specific activity is lower if the cells have been exposed to high NaCl or urea. Further, high salt reduces Gdpd5 mRNA abundance and a specific siRNA against Gdpd5 increases GPC. Thus, Gdpd5 is a GPC-PDE, whose activity contributes to osmotic regulation of GPC. High NaCl or urea decreases phophorylation of Gdpd5, which reduces its activity. We have identified several more signaling molecules that regulate TonEBP activity. c-Jun and c-Fos bind to an AP-1 site adjacent to the OREs in many genes that are regulated by TonEBP, and presence of the site and of c-fos and c-Jun activity are necessary for full activation of TonEBP by high salt. High NaCl activates c-Abl, resulting in increased phosphorylation of TonEBP-Y143. High NaCl also inhibits SHP-1 phophatase, which contributes to phosphorylation of TonEBP-Y143. When Y143 is phosphorylated, phospholipase C gamma binds to TonEBP, increasing its transcriptional activity, nuclear localization, and transactivating activity. Finally, we previously found that hyperosmolality causes DNA breaks and oxidative stress both in cell culture and in kidney medullas in vivo. DNA damage and oxidative stress are associated with cellular senescence, most striking in aging and in cancer. We have now found that high salt causes cellular senescence in tissue culture and that age-associated accumulation of a senescent cells is accelerated in kidney medullas of normal mice, as well as in C. Elegans exposed to high salt. Thus, hyperosmolality not only causes DNA damage and oxidative stress, but also causes cellular senescence.